We know very little about the molecular mechanisms underlying the organization of cell-fates and circuitry in the vertebrate midbrain. The midbrain contains important nuclei (dopaminergic, oculomotor and red nuclei) that govern addictive behaviors, eye, limb and voluntary movements. I have shown that during development, these nuclei emerge from stripes of cell-fates (arcs) specified by a source of Sonic Hedgehog (SHH). But my recent findings suggest an as yet unexplored hypothesis: that though sufficient, SHH is not necessary for midbrain specification and that multiple hedgehogs (SHH. Indian, Desert) derived from neural (chick) or extraneural sources (mouse, chick) could jointly pattern the ventral midbrain. How is the multi-ligand HH signal transduced to produce specific midbrain cell-fates? I examine this issue at the level of the HH ligands (Aims 1 and 2) and the final and essential effectors of the HH cascade: the Gli genes (Aim 3). Gli 1,2 and 3 carve out 3 discrete progenitor domains in the midbrain, possibly reflecting a mechanism by which distinct midbrain cell-fates could be specified. Although Shh:Gli gene interactions have been studied in the spinal cord, these studies do not elucidate how important midbrain nuclei (e.g. dopaminergic) develop. The aim of this proposal therefore is to investigate the interactions between multiple HH and multiple Gli genes in specifying midbrain cell-fates. The first aim will explore the role of individual HH genes and all HH signaling by gene expression analyses of the shh-/-, Indian Hedgehog (ihh-/-), shh:ihh-/- and smoothened (smo-/-) mice (where all HH signal is lost). The second aim will determine whether SHH is necessary or if other HH genes are involved in midbrain patterning in acute loss of function experiments in chicks and mice. All HH gene function will be blocked by electroporating negative regulators of HH signaling (Ptc-delta-loop2, HHIP). Next, individual Hh gene function will be blocked in chicks and mice by electroporation of RNA interference (RNAi) constructs. The third aim will examine the role of Gli genes in midbrain patterning. Gli genes will be overexpressed and knocked down (RNAi) in the discrete midbrain domains delineated by Gli gene expression. SHH-Gli gene interactions will be analyzed in the shh-/-, smo-/- and gli3 -/- single and double knockouts by gene expression and the misexpression and knockdown of HH pathway genes in organotypic explants. Finally, midbrain and spinal cord defects in the chick Talpid2 mutant, where the SHH:GLI3 ratio and lateral floor plate specification are perturbed, will be characterized by gene expression. Attempts will be made to rescue the Ta2 mutant by misexpression of candidate Hh pathway genes (SHH, GLI3, DZIP1, HHIP; PTC1, SCUBE2). Significance: A study of the HH-GLI pathway is critical for understanding how the cell fates and circuitry of midbrain neurons, which govern motor and addictive behaviors, are determined. Understanding the specification of midbrain neurons is also crucial to the development of stem cell based therapeutic strategies to combat Parkinson's disease and stroke.